EP1095967A1 - Novel cell culture supports carrying special properties and their production - Google Patents

Abstract

Production of 2-dimensional supports for anchorage-dependent cell culture involves activating polymer film to generate reactive groups (e.g. peroxide, hydroperoxide or amino) and then grafting with a polymer or macromolecule of interest by immersing in monomer solution and polymerizing with free radicals formed by irradiation with beta -rays. A process for the continuous production of 2-dimensional supports with special functions for the culture of anchorage-dependent cells (CAD) comprises: (1) continuous production of reels of polymer film with a thickness of up to 35 microns and a density of 0.9-1.25 g/cm<3> from polymer granules; (2) activation by generating reactive groups, especially peroxide, hydroperoxide or amino groups; (3) covalent grafting between the activated polymer and a (co)polymer or macromolecule of interest by immersing the film in a monomer solution and polymerizing with free radicals created by activation with beta -radiation, the immersion time being directly correlated with the required thickness of grafted polymer; (4) if necessary, washing to remove unreacted and unattached monomer, followed by drying and (5) cutting to the required size by continuous stamping. Independent claims are also included for the following: (i) two-dimensional micro-supports for bulk CAD culture, made by this process and comprising a base polymer film with a thickness of 10-35 microns and a grafted layer with a thickness of 1-10 nm; (ii) apparatus for the above process comprising: (i) a system for unwinding/winding the polymer film at a speed selected for each of the stages of activation, grafting and punching out; (ii) an electron accelerator suitable for the continuous activation of reels of film; (iii) a container for monomer solution and for the grafting polymer or macromolecule, through which the film passes continuously at a speed determined by the winding/unwinding system; and (iv) a tool for punching out the film as it unwinds.

Description

The present invention relates to a new process for obtaining two-dimensional (2D) microcarriers for the culture of anchor-dependent cells. More specifically, the invention relates to a method of preparation of such microcarriers suitable for mass culture, in infusion or in clusters of anchor-dependent animal cells and exhibiting particular properties such as cryosensitivity, biocompatibility, biodegradability or specific adhesion. The invention also relates to the microcarriers obtained by this process and their use. Finally, it relates to a device which makes it possible to manufacture these microsupports in mass, size and homogeneous constitution.

Anchor-dependent cells (CAD) are dependent on adherence to a medium to proliferate and preserve their functions and their viability. This dependence is a bottleneck technological bottleneck for the production of substances biological and pharmaceutical products secreted by these CAD in comparison with those from anchor-independent cells which can proliferate in suspension. This dependence comes in particular from the fact that the CAD growth stops when they reach confluence and that it is necessary to detach the confluent cells by trypsinization. In addition, the need for cells in nutrient medium and oxygen limits the number and / or the surface of the microcarriers for a given volume of culture medium. Different cropping systems have been developed in order to present a sufficient anchoring surface only for allow CAD to be produced on an industrial scale: "roller bottles", "multi-tray" systems, hollow fibers, and microcarriers, particularly interesting in terms of surface / volume ratio.

Many microsupports used industrially to mass culture anchor-dependent cells (CAD) are characterized by three-dimensional geometry (3D) and the easiest to shape are spherical geometry. Used by the biopharmaceutical industry to make cell culture (bioreactor up to 1000 L) in batch mode, 3D microbeads, an example of which is Cytodex ^R sold by Pharmacia (UPPSALA, Sweden) suspended at 5g / l allow to obtain concentrations of the order of 2.10 ⁶ cells / ml, 7% of the culture volume being occupied by the microbeads. These constitute a reference in the field. However, the surface / volume ratio of spherical microcarriers is not favorable to an increase in the concentration of microcarriers in the bioreactor. If you want to cultivate CAD at high concentrations (10 ⁷ to 5.10 ⁷ cells / ml), you must be able to increase the concentration of microsupport in the bioreactor. However, there comes quickly a moment when the volume of the swollen microbeads represents an excessive proportion of the culture volume, reducing the volume of medium available for the cells.

A new generation of microsupports with two-dimensional geometry was developed and described in EP 579 596.

By two-dimensional geometry, it should be understood that the thickness of these microcarriers tend to become tiny and negligible compared to dimensions of cultured cells. This reduction in thickness is such that it there is no possibility of cell growth, nor within the support, nor on its edge but only on its two anchoring faces. These 2D microsupports (2D-MS) offer the main advantage of a surface anchor per unit of volume higher than all 3D competitors, such as the CYTODEX® microbeads mentioned above.

They therefore make it possible, for an occupation of culture volume given in a bioreactor, to cultivate and obtain more cells per unit of volume. Indeed, the external anchoring surface of a sphere (4πR ² ) is equal to the total surface of two infinitely thin discs (2 x 2πR ² ) located at the equator and forming part of this sphere. However, the combined volumes of these two "equatorial" discs become smaller the smaller the thickness of the film used to produce them, representing only a tiny fraction of the volume of the sphere which circumscribes them. Adopting a thickness of 10 µm for all of the disks generated in a sphere, the total surface capable of anchoring the cells thus provided by the disks is approximately 3.3 times greater than the outer surface of the sphere, taking into account the random movements of these, in suspension.

The large anchoring surface available for CAD per unit of volume of 2D-MS therefore makes it possible to envisage the culture of cells with high industrial scale concentrations.

Another limitation of supports for anchor-dependent cell culture is that of the recovery mode of cells attached to their support while preserving the biological properties or physiological of the latter. Indeed, the detachment of cells from their support goes through an enzymatic treatment (such as trypsin) or a chelating agent (such as EDTA), which can damage not only cellular functions but also their subsequent re-attachment to supports in the context of continuous cultures. This limitation is particularly detrimental when the biological functions of cells are then used industrially. This situation is frequently encountered in the case of production of macromolecules of interest by said cells, or when all of the receptors or molecules membranes is sought after for their ability to bind ligands or internalize molecules or substances.

In addition, culture at confluence of anchor-dependent cells leads to the formation of intercellular links or junctions intercellular. These intercellular junctions also contribute to the functionality of cells and their destruction at the loss of these features.

These limitations in mass culture of anchor-dependent cells also lead to a limit to industrial production of biological macromolecules produced by these cells which it these are molecules normally synthesized by cells in question or more generally produced by insertion by techniques of genetic recombination of genes coding for a protein heterologous. This limitation linked to cell production capacities anchoring-dependent functional can lead to cost prices manufacturers of proteins that we wish to express and purify, incompatible with a subsequent sale price of a medicinal product containing said protein as active ingredient.

• haut rendement de cellules par unité de volume de milieu de culture ;
• altération minimale de la viabilité des CAD consécutivement à leur décrochage des microsupports ;
• contrôle du nombre et de la spécificité des cellules susceptibles de s'ancrer sur lesdits supports, ledit contrôle permettant d'adapter ces supports, d'une part, au système de culture utilisé, d'autre part, aux fonctionnalités desdites cellules cultivées ;
• possibilité de produire à grande échelle ces microsupports pour cellules ancrage-dépendantes avec une excellente reproductibilité et à un prix de revient industriel compatible avec le prix de revient de production de cellules ou de macromolécules d'intérêt biologique.

There is therefore a real need for microcarriers for the culture of CAD having the following specificities:

• high cell yield per unit volume of culture medium;
• minimal deterioration of the viability of CAD following their detachment from microcarriers;
• control of the number and specificity of cells capable of being anchored on said supports, said control making it possible to adapt these supports, on the one hand, to the culture system used, on the other hand, to the functionalities of said cultured cells;
• possibility of large-scale production of these microsupports for anchor-dependent cells with excellent reproducibility and at an industrial cost price compatible with the production cost of cells or macromolecules of biological interest.

Attempts to overcome a number of disadvantages described above, including the need to use enzymes or chelating agents to unhook cells from their support, have been described in EP 387 975 and in EP 382 214. These two patents propose to cover conventional cell culture supports with the product of the copolymerization of hydrophilic monomers chosen from poly-N-alkyl- (meth) acrylamide derivatives, their respective copolymers, the poly-N-acryloyl-piperidine or poly-N-acryloyl-pyrrolidine, the functionality sought is cryosensitivity.

In this use, the polymer is chosen according to the critical lower temperature or LCST which is a temperature of transition for hydration and dehydration of the polymeric compound. When the LCST is below the cell culture temperature, the cells remain fixed on the polymer support during the cellular culture. They can be taken down after a lowering the temperature of the culture such that the latter is substantially lower than the LCST. The methods of obtaining a polymer or copolymer with a lower LCST temperature data are described in EP 382 214 B1. All polymers grafted from the monomers cited in this patent application are suitable but not limited in application to cryosensitivity herein request. In general, it can be said that the inclusion of monomers hydrophilic in the polymerization process tends to increase the LCST, while the presence of hydrophobic polymers tends to decrease the LCST. Examples of hydrophilic monomers are: N-vinyl-pyrrolidone, vinylpyridine, acrylamide, methacrylamide, N-methyl-acrylamide, hydroxyethyl-methacrylate, hydroxyethyl-acrylate, hydroxymethyl-methacrylate, hydroxymethyl-acrylate, acrylic acid and methacrylic acid having acid groups and their salts, vinylsulfonic acid, acid styrylsulfonic and N, N'-dimethylamino-ethyl-methacrylate, N, N'-diethylamino-ethyl-methacrylate, and N, N'-dimethylamino-propyl-acrylamide having basic groups and their salts.

Examples of hydrophobic monomers are: derivatives acrylates and methacrylate derivatives such as ethyl acrylate, methylmethacrylate and glycidyl-methacrylate etc., N-substituted-alkyl (meth) -acrylamide derivatives such as N-nbutyl- (meth) -acrylamide and N-isopropyl-acrylamide etc. as well as vinyl chloride, acrylonitrile, styrene, and vinyl acetate etc.

• ni le contrôle de la quantité et donc de l'épaisseur des polymères greffés, paramètre particulièrement important lorsque l'on veut contrôler la densité des cellules en culture ;
• ni d'assurer un couplage covalent du polymère ou copolymère sur le support de culture ; ceci est un inconvénient majeur dans la mesure où il ne permet pas la conservation à long terme de ces supports ni leur réutilisation.

However, the means described for coupling these cryosensitive polymers or copolymers to the cell culture supports do not allow:

• nor the control of the quantity and therefore of the thickness of the grafted polymers, a particularly important parameter when it is desired to control the density of the cells in culture;
• nor to ensure a covalent coupling of the polymer or copolymer on the culture support; this is a major drawback insofar as it does not allow long-term preservation of these supports nor their reuse.

• une production en continu de bobines de film polymère d'une épaisseur inférieure ou égale à 35 µ à partir de granules d'un polymère donné ;
• une activation dudit film polymère par tout moyen permettant de générer des groupements réactifs notamment des radicaux ou des fonctions peroxydes, hydroperoxydes ou amines ;
• un greffage covalent entre le film polymère activé et un polymère, un copolymère ou une macromolécule d'intérêt dont la fonctionnalité est recherchée, ledit greffage étant obtenu par immersion du film dans une solution de monomère, la copolymérisation étant amorcée par les radicaux libres créés par l'activation sous rayonnement β, le temps d'immersion étant directement corrélé à l'épaisseur recherchée du polymère greffé sur le film ;
• le cas échéant un lavage pour éliminer les monomères non consommés et non fixés sur le film ;
• une découpe du film polymère par un procédé choisi en fonction de la géométrie et de la taille des microsupports 2D recherchées ;
• le cas échéant, une étape de stérilisation, soit par autoclavage, soit par rayonnement, la méthode de stérilisation étant bien entendu choisie en fonction du matériau à stériliser. Dans le cas où le matériel n'est pas autoclavable, il doit être stérilisé par une méthode physique (rayonnement γ ou β) ou chimique (mélange isopropanol/H₂O 70/30 % (v/v)).

The present invention relates to a process for continuously obtaining two-dimensional (2D) microcarriers exhibiting particular functionalities for the culture of anchor-dependent cells (CAD). It is illustrated in FIG. 1 and comprises at least the following sequence of steps:

• continuous production of reels of polymer film with a thickness of 35 µm or less from granules of a given polymer;
• activation of said polymer film by any means making it possible to generate reactive groups, in particular radicals or peroxide, hydroperoxide or amine functions;
• a covalent grafting between the activated polymer film and a polymer, a copolymer or a macromolecule of interest whose functionality is sought, said grafting being obtained by immersion of the film in a monomer solution, the copolymerization being initiated by the free radicals created by activation under β radiation, the immersion time being directly correlated to the desired thickness of the polymer grafted onto the film;
• if necessary, washing to remove the monomers not consumed and not fixed on the film;
• cutting the polymer film by a process chosen according to the geometry and the size of the 2D microsupports sought;
• if necessary, a sterilization step, either by autoclaving or by radiation, the sterilization method being of course chosen as a function of the material to be sterilized. If the material is not autoclavable, it must be sterilized by a physical (γ or β radiation) or chemical (isopropanol / H ₂ O 70/30% (v / v)) mixture.

A particularly advantageous process includes cutting by continuous punching, the size of the punches being that of the size 2D microsupports sought.

It is the combination of these different stages including a activation followed by covalent grafting of a polymer film (substrate) previously implemented and then cutting said carrier substrate film of the polymer, copolymer or macromolecule grafted covalently by a process for obtaining homogeneous 2D microcarriers in size and geometry which makes all the originality of the invention.

• de densité comprise entre 0.9 et 1.25g/cm³ et de préférence entre 1 et 1.1 g/cm³ pour permettre une culture agitée en suspension dans du milieu de culture (en bioréacteur), sans risque de sédimentation ou de flottation.
• transparent pour permettre une analyse aisée des cellules au cours de leur croissance, en continu dans le bioréacteur. Par transparent, on entend que dans des longueurs d'ondes comprises entre 400 nm et 1000 nm, la lumière traverse les microporteurs sans atténuation importante de l'intensité du faisceau lumineux émergeant par rapport à l'intensité du faisceau de lumière incident. Une adsorption inférieure à 1 % est considérée comme tout à fait avantageuse ;
• caractérisé par une balance hydrophobe/hydrophile adéquate : s'il est plutôt hydrophobe, il doit avoir un angle de contact tel qu'il soit suffisamment mouillable en milieu aqueux et s'il est plutôt hydrophile, il ne doit pas gonfler dans l'eau ; autrement dit, l'angle de contact  entre la surface du matériau et une goutte de milieu aqueux doit être compris entre 30° et 90°, l'angle de contact étant défini comme l'angle formé entre la surface du matériau et la tangente de la goutte au point triple d'intersection entre la goutte, la surface du matériau et l'air.

As regards the raw material used in the form of a film, the nature of the polymer cannot be arbitrary. It is desirable according to the invention for the material to be:

• density between 0.9 and 1.25 g / cm ³ and preferably between 1 and 1.1 g / cm ³ to allow a stirred culture in suspension in the culture medium (in bioreactor), without risk of sedimentation or flotation.
• transparent to allow easy analysis of cells during their growth, continuously in the bioreactor. By transparent, it is meant that in wavelengths between 400 nm and 1000 nm, the light passes through the microcarriers without significant attenuation of the intensity of the emerging light beam relative to the intensity of the incident light beam. An adsorption of less than 1% is considered to be entirely advantageous;
• characterized by an adequate hydrophobic / hydrophilic balance: if it is rather hydrophobic, it must have a contact angle such that it is sufficiently wettable in an aqueous medium and if it is rather hydrophilic, it must not swell in water ; in other words, the contact angle  between the surface of the material and a drop of aqueous medium must be between 30 ° and 90 °, the contact angle being defined as the angle formed between the surface of the material and the tangent from the drop to the triple point of intersection between the drop, the surface of the material and the air.

An angle  = 0 corresponds to perfect wetting and the surface of liquid is parallel to the surface of the material. An angle > 90% corresponds to a situation of no wetting and drops remain formed on the surface of the material. A contact angle  between 30 and 90 ° corresponds to an imperfect wetting corresponding to a spread partial drop on the material.

According to the invention, the polymer material which can be used for producing the films (substrate) can thus be polystyrene, polyethylene, polyethylene terephthalate or polycarbonate or any copolymer mainly including these rather hydrophobic polymer materials. In as a more hydrophilic film, the material used can also be cellophane or aliphatic polyester of the polylactide type or polyhydroxybutyrate and any copolymer mainly including these more hydrophilic materials.

When the nature of the polymer used is of polyester type aliphatic, the film is in essence bioresorbable / biodegradable and, in in this case it can be used as an implant in the living organism if the polymer used is preferably recognized biomedical grade by the FDA.

Preferably, the thickness of the film used to produce the microsupports is between 10 and 25µ.

Continuous production of reels of thin polymer film or ultra-thin is produced by the "extrusion-stretching" technique from granules of a given polymer. This raw material is heated to reach the molten state, then extruded through a rotating screw and molded between two plates to produce a thick polymer film. To the leaving the extruder, the fairly thick film is then stretched hot, either in one direction, either in two directions orthogonal to each other to then produce a non-shrink film whose thickness is much smaller and customizable (between 10 and 35µ) over the entire width of the coil produced, this within the limits of thermal properties and mechanical of the raw material.

The "blow-molding" technique is an alternative continuous film work. The variant is located at the second step, namely, the injection of air between two walls of films which has the role to extend the material and therefore to obtain a film thickness more low. Other techniques for processing thin films are described in other applications of polymer chemistry (such as biosensors): "spin-coating" or "solvent-casting" but these two techniques are more often used to make sheets or discs of small size and are less suitable for producing continuous film reels.

In a second step, the polymer film, which can advantageously be in a rollable form with a width between 5 cm and 60 cm and a length of at least 3 km, undergoes then an activation in order to generate groupings reagents, capable of forming covalent bonds with others reactive groups of the substance that one wishes to graft. In this meaning, by substance, is meant organic monomers or polymers having particular properties, in particular properties of cryosensitivity or biocompatibility; it can also be biological macromolecules which have a specific affinity for certain cellular receptors: 2D microcarriers resulting from such grafting then allow the selective adhesion of certain types of cells present in an initial cell sample comprising a mixture of cells. By way of example, mention may be made of skin cells (keratinocytes) having reached different stages of differentiation, or cells resulting from the insertion, activation or repression of a particular function, in particular by inserting a gene carrying said function or carrier of a regulatory function for gene expression cellular.

• la nature des groupes chimiques induits dans le polymère par l'activation,
• la profondeur de traitement dans l'épaisseur du matériau.

Four methods are used to modify the chemical structure of polymers and generate reactive groups. These are electron beams and, more particularly, β radiation, crown or corona discharges, UV treatment and, finally, plasmas. For each process, two parameters will guide the choice of method according to the properties sought for the material subjected to this radiation:

• the nature of the chemical groups induced in the polymer by the activation,
• the depth of treatment in the thickness of the material.

In all cases, activation consists in subjecting the support to a electromagnetic radiation which breaks the bonds and the creation of free radicals, peroxide, hydroperoxide functions or amines.

If necessary, and according to methods already described, molecules spacers can be grafted if desired via free radicals thus generated. Their function is to increase the length of the link between reactive sites and the monomers, polymers or macromolecules that are wants to covalently bond to the polymer film, and therefore to increase their mobility.

Activation consists in subjecting said film to a bombardment electronic. This bombardment is preferably carried out under inert atmosphere. In the process of the invention, it is essential that the activation step precedes the grafting step. When these two stages are simultaneous as in the case of patent EP 382,214, the monomer which one wishes to graft is then also subjected to radiation and a very large amount of homopolymers is created free in solution which can adsorb on the surface of the support and which must be then wash off. In this case, it is then difficult to ensure that all the ungrafted free chains are eliminated by washing, and that these free adsorbed chains do not dissolve in the middle of culture during cell detachment by thermal contrast.

• la nature du film polymère à greffer ;
• la nature du copolymère ou des macromolécules que l'on souhaite coupler de façon covalente au film polymère ;
• le caractère discontinu ou continu du procédé d'activation : le support greffé qui sera ensuite découpé peut en effet être traité de façon statique ou en continu par défilement du film.

The activation conditions are chosen according to a certain number of parameters among which appear at least:

• the nature of the polymer film to be grafted;
• the nature of the copolymer or macromolecules which it is desired to couple covalently to the polymer film;
• the discontinuous or continuous nature of the activation process: the grafted support which will then be cut can indeed be treated statically or continuously by scrolling the film.

When the activation process is continuous, the scrolling speed of the film can vary from 0.1 to 10 m per minute and be established according to the total radiation dose required to activate the film and the power of the irradiator fixed according to the thermal resistance of the film.

In the case of irradiation of polystyrene film by β radiation or γ, we can increase the power of the irradiator up to 6 milliamps (my) ; beyond, there is overheating and deformation of the film. If we decrease appreciably the speed of movement of the film, with an intensity fixed at 6 mA, doses of 80-200 kg maximum can be reached in one only one pass under the irradiator.

The Kilogray or joule per gram is a representative unit of the dose and depends on the characteristics of the electron beam unit.

When a corona discharge is used, it is emitted at a voltage of several thousand volts and at frequencies the located in the kHz domain. This process is used in an atmosphere ambient. The geometric amplitude of the corona arc is a few millimeters for most conventional systems to a few centimeters for blown arc systems. The discharge is carried out with parallel electrodes located on either side of the object. The use of crown discharges for film activation has the advantage of being a treatment under ambient atmosphere.

Activation of the polymer film by cold plasma is carried out also using electrodes which emit discharges into the radio frequency domain.

A plasma is obtained by ionization using a high source frequency of a gas or a mixture of gases introduced into an enclosure put under a residual pressure of a few millibars. This process expensive is also difficult to implement continuously on a film polymer. However, these three types of activation: electron beam, β or γ, crown or plasma discharges are suitable for generating reactive groups.

According to the invention, the technique of radiografting of monomers of acrylamide or vinyl type as described above on the surface of a thin polymer film of polystyrene or aliphatic polyester type must therefore be initiated by radiation whatever the nature of it. The grafting step is then carried out directly by immersion in immersing the pre-activated polymer film in a solution of the monomer, of a mixture of several selected monomers or macromolecules.

If it is an organic monomer, its copolymerization with surface of the polymer film is initiated by free radicals created during pre-irradiation, and the polymer chains formed are linked by covalent to the film. The reaction is instantaneous, but, depending on the nature of the film, it can last for a few seconds or even a few minutes to arrive at a maximum grafting rate. This grafting rate is directly related to the irradiation dose, the concentration of monomers in the impregnation bath, and at the time of the reaction.

The method according to the invention thus comprises inter alia optimization of the four parameters mentioned above, namely: nature and irradiation dose, grafting time, concentration of monomers and nature of the solvent in the grafting bath, in order to obtain a layer of polymer, copolymer or specific macromolecules grafted so covalent and desired thickness. In fact, as shown in Example 2 below, the thickness of the layer of polymer, of organic copolymer, or specific macromolecules deposited is determined by analysis "X-ray photon spectroscopy" (XPS) and directly depends on the duration of the grafting step by impregnation in the bath.

In the case where the pre-irradiation step is carried out in air, the reactive groups created are capable of being oxidized instantly in the presence of air. In this case, the grafting step is carried out extemporaneously by immersion in a bath containing an ad hoc compound which makes it possible to regenerate the reactive radicals.

In the process of the invention, the grafting step can be the case followed by a washing step which consists in removing the residues from the biological monomer, polymer or macromolecules that have not been consumed and / or polymerized on the surface of the polymer film. These rinses are generally carried out in a mixture of isopropanol in water (70/30% v / v) until there is no longer any trace of reagents and / or products in the washing phases. In the application to culture of cells, it is essential that washing is as efficient as possible made of the cytotoxicity of acrylamide monomers or polymers and derivatives.

In the process of the invention, different natures of polymers, copolymers or macromolecules can thus constitute a layer of between 1 and 10 nanometers on which the adherent cells attach and proliferate. When total or partial cryosensitivity is sought, the polymer or copolymer of interest is chosen from poly-N-alkyl (meth) acrylamide derivatives, their respective copolymers, poly-N-acryloyl-piperidine or poly-N -acryloyl-pyrrolidine. When biocompatibility is sought, surface treatment involves, for example, covalent grafting of hydrophilic polymer of the polyethylene ethylene oxide PEO type on the surface of a polymer film pre-exposed to an allylamine plasma to generate amine functions. surface, and this by means of a suitable chemical coupling agent, such as for example cyanide chloride. This type of treatment is described in (J. Biomed. Mater. Res. 1991, 25 , 1547).

When the property sought is a selective adhesion of animal cells, the conventional methods of grafting macromolecules onto the supports as described in particular in the techniques of affinity chromatography using antibodies, aptamers or molecules obtained by combinatorial chemistry, can be used . Those skilled in the art can refer, for a detailed description of these techniques, to (J. Cell. Biol. 1991, 114 , 1089 § 1990, 110 , 777, J. Biol. Chem. 1992, 267 , 14019 § 10133 , Art. Organs 1992, 16 , 526, Macromolecules, 1993, 26 , 1483). The biological macromolecules (oligopeptides, oligonucleotides etc.), which can advantageously be grafted onto the supports prepared by a method according to the invention, are ligands specific for cellular receptors making it possible to achieve the growth of a certain type of cells in a culture medium at the expense of other cell types which could be mixed with it. Even more particularly, this could make it possible to multiply the cells which express, either naturally, or resulting from an in vitro genetic recombination of these cells expressing a specific macromolecule at the level of their membrane.

In the process of the invention, the substrate polymer film on which have been grafted a polymer, a copolymer or a macromolecule of interest, is then cut by a process chosen according to the geometry and size of the desired 2D microcarriers.

A preferred implementation in the present invention is the punching, the size of the punches being that of the 2D micro-supports products.

According to a preferred embodiment of the invention, the microcarriers have a thickness preferably less than or equal to 25 µ as well as a disc shape. The last step in the production process such grafted 2D-MS therefore includes a cutting of the grafted film thin or ultra-thin polymer (substrate) in micrometric particles characterized by two-dimensional geometry and comprising two anchor faces on each of which the cells hang and proliferate without possible penetration of cells between the two faces.

In our process, we have selected a technique of punching cutting which provides excellent quality of microdisks produced, in terms of homogeneity of size, shape and thickness of the microdisks supplied, and in terms of absence of debris.

Homogeneity of size (homodispersity) is essential to the synchrony of the different stages of cell growth. Indeed, the presence of microcarriers of various sizes would imply that of small ones would reach confluence before those of large size. In this case, the CAD which reached the confluence earlier, can partly drop, die and release ammonia, lactic acid and others toxins that are deleterious to the growth of CAD proliferating on larger microcarriers.

This step is carried out by punching the polymer film only pre-activated or pre-activated then grafted with circular punches of selected diameter (for example 150 μ) in ceramic carbide. The film polymer is punched at a cutting frequency (beats by minute) optimized as a function of the speed of advancement of the polymer film graft.

Other cutting techniques can be envisaged. Of a hand, the embossing systems using a rotary knife consisting of engravings inserted on a printing cylinder of given diameter, a second cylinder of the same diameter serving as a press or "imprint". There is has a number of punch lines given on the 360 ° of the cylinder (defined depending on the spacing between punches and the cylinder diameter) and a number of punches per line given (defined according to the length of the cylinder). The cutting speed of a reel of film passing between the two cylinders and therefore the productivity must be different from our process. It is a technique widely used by companies that market labels of all sizes and shapes. In this case, the pressure it would take applying for cutting may limit the process.

The invention also relates to microcarriers with two dimensions (2D) endowed with particular functionalities for the culture of dependent anchor cells (CAD), as obtained by a described process above, characterized in that the thickness of the polymer film is included between 10 and 35 µ, and that of the polymer, the copolymer or a macromolecule of interest covalently grafted have a thickness between 1 and 10 nm.

The succession of stages in the fabrication of grafted 2D-MS is given in figure 1.

When 2D-MS are cryosensitive, a minimum thickness of 5 nanometers for the grafted layer is necessary if one wishes that confluent cells are quantitatively detached from their support. Thinner thicknesses are sought when wish to detach partially and not quantitatively by contrast thermal of CAD at confluence.

• un système de déroulement/enroulement du film polymère, l'entraínement du film étant effectué à une vitesse choisie au niveau de chacune des étapes : activation, greffage, poinçonnage ;
• un irradiateur permettant d'activer la surface du film ;
• un bain de greffage destiné à contenir la solution de monomères, polymères ou macromolécules à greffer, dans lequel le film polymère préactivé passe, de façon continue, à une vitesse déterminée, entraíné par le système d'enroulement/déroulement ;
• un outil de découpe permettant de réaliser le poinçonnage du film lors de son déroulement.

The invention also relates to a device for the continuous preparation of 2D-MS support endowed with particular properties, prepared according to a process as described above and using a covalent attachment of a polymer, copolymer or biological macromolecules to a substrate made of a polymer film, said device comprising:

• a system for unwinding / winding the polymer film, the film being driven at a speed chosen at each of the stages: activation, grafting, punching;
• an irradiator for activating the surface of the film;
• a grafting bath intended to contain the solution of monomers, polymers or macromolecules to be grafted, in which the pre-activated polymer film passes continuously, at a determined speed, driven by the winding / unwinding system;
• a cutting tool allowing the punching of the film during its unwinding.

FIG. 2 represents the diagram of the device of the step of continuous film cutting (in the form of reels). The system rewinder-unwinder serves as a supply and evacuation tool for cutting. In addition, this tool will operate 24 hours a day. M1 is the engine governing the tape feed, M2 is the reel winding motor and M3 is the reel unwinding motor. C1 represents the sensor to start M3, C2 is the stop sensor for M3, C3 represents the M2 and C4 start sensor is the M2 stop sensor. Finally, R1 indicates the band braking system.

Preferably, the film unwinding / rewinding system allows the advancement of said film at a speed of between 0.1 and 8 m by minute. The unwinding / winding speed is much slower at cutting stage only during the activation and grafting stages.

The heart of the device consists of a tool block on which are inserted, along the length of the film, rows of punches circulars and the corresponding imprint block. For example, for a 5 cm wide film, the tool block will include 9 rows of 50 punches each. This configuration of the online tool block in the direction of length rather than width increases maintenance security. On the same line of punches, the spacing between them is 0.25 mm; the regular pitch between 2 lines is 0.20 mm. It is clear that the arrangement cited above, the diameter of the punches and their spacing is an optimal proposition but can of course be adapted in according to needs and has no binding character.

As punching takes place, the 2D-MS grafted microcarriers are then continuously collected. By way of example, from a spool of polystyrene grafted with a width of 5 cm and a length of 3 km, the device according to the invention comprising punches with a diameter of 150 μ makes it possible to obtain approximately 1 kg of confetti (2D-MS microsupports) 150 µ in diameter. Knowing that the surface density of the material is 26.25 g / m ² , the minimum number of particles obtained equals 2.38 10 ⁹ microcarriers per coil or per kilo of microcarriers produced. In the configuration cited above by way of example, the cutting yield is 28%.

FIG. 3 represents the 2D-MS microcarriers thus obtained.

Microdisks can be collected in receptacles positioned directly under the punching die.

The examples below allow, without limiting them, to show the advantages of the cell culture method and supports such that obtained by this process.

Example 1 - Radiografting using the pre-irradiation technique using electron beam under nitrogen:

First, we sought to optimize and define the parameters of discontinuous radio-grafting (total activation dose, nature of the solvent, concentration of the monomer, temperature of the grafting bath, grafting time) on sheets ( 5 x 10 cm ² polystyrene film 25 µ thick) with a Van der Graaf type static research electron accelerator, characterized by low power and low dose rate compared to industrial devices. These leaves are placed beforehand in 75 cm ² culture flasks, pre-degassed and irradiated to dryness under an inert atmosphere (nitrogen) as follows:

The polystyrene culture flasks containing the polystyrene film are washed twice in an Isopropanol / H ₂ O 70/30% (v / v) mixture and dried under a stream of nitrogen for 15 minutes.

The previously degassed bottles are placed under an irradiator High energy static EB (10 Mev). An electron accelerator (10 MeV) Van der Graaf type, the intensity of which is fixed at 1 mA and the dose rate set at 10 Kgray / min (1 Mrad / min), is used. The bottles are therefore irradiated under the beam for a fixed time (in minutes) so to absorb a given total dose (in Kgray or Joule / g). The dose deposited is calibrated beforehand with a dosimeter.

At the outlet of the irradiator, the bottles are immediately brought into contact with the solution (H ₂ O), stock of monomer (concentration by weight of 10 to 40%) under a nitrogen atmosphere. The freshly prepared stock solution of monomer is transferred to the polystyrene bottle pre-irradiated by a nitrogen push system. The grafting solution, once transferred to the pre-irradiated bottle, is balanced at a given temperature (from 25 ° C to 60 ° C). The grafting time (from 0.5 to 24 hours) is varied at a given temperature in order to check the influence of various parameters on the thickness of the layer of Poly-N-isopropylacrylamide on the surface of the film like the culture flask in polystyrene. The grafting solutions are removed from the vials after a given time and then the vials and the grafted films are washed three times and then dried.

Example 2 - grafting of N-lsoPropylAcrylAmide (NIPAAm) on a polystyrene film:

2.1 Study of the thickness of the deposit as a function of the reaction time: Films 25 microns thick are irradiated under nitrogen using an electron accelerator of the Van der Graaf type (10 Mev) and a total dose of 250 Kgray . The irradiated films are immediately immersed in an aqueous solution of NIPAAm (10% by weight) which has been degassed beforehand for 30 min with nitrogen. The temperature of the grafting solution is 60 ° C. Different grafted surfaces have been obtained by varying the concentration of monomer in the grafting solution and the reaction time. When the grafting reactions are finished, the grafted films are washed, dried and analyzed by "X-ray Photon Spectroscopy" (XPS), in order to determine the chemical composition of the extreme surface. The thickness of the deposit as a function of the time of reaction is shown in Table 1 below. Films grafted according to Example 1 - XPS analysis Film samples (Grafting time) % Atomic Experiment. Deposition thickness (in A) % cell dropout (by counting) VS O NOT Native polystyrene 98.5% 1.5% 0% - - Grafted polystyrene (1/2 h) 92.3% 4.1% 3.6% 38 Å 65.5% Grafted polystyrene (1 h) 78.7% 11.3% 10% 47 Å 79.0% Grafted polystyrene (3 h) 75.7% 13.1% 11.2% > 50-60 Å 96.5% Native Poly-N-IsoPropyl-AcrylAmide 75.0% 12.7% 12.3% - - The C _1S peak of the carbon of the native polystyrene can be broken down into two components (at 284.8 eV and 291.5 eV) which correspond respectively to carbon involved in hydrocarbon bonds and to the “shake-up” peak characteristic of aromatic compounds. According to the literature, this last peak has an intensity of 7% relative to the total carbon for the polystyrene. The thickness of the film was calculated by the surface ratio (%) of the peak C _1S “shake up” characteristic of the polystyrene and of the total C _1S peak for the grafted film sample analyzed at an electron collection angle of 90 ° (Quantitative analysis). In the case where the peak C _1S “Shake up” characteristic of pure polystyrene, is no longer detected in XPS, the thickness of the grafted deposit is greater than the analysis depth of the XPS technique, namely 50 to 60 Å .

2.2 Films of 25 µm thickness are irradiated discontinuously under nitrogen using a Van der Graaf type electron accelerator (10 Mev) and a total dose of 150 Kgray. The irradiated films are immediately immersed in an isopropanol solution of NIPAAm (10% by weight) which has been degassed beforehand for 30 minutes with nitrogen for 3 hours. The temperature of the grafting solution is 20 ° C. No deposition of polyN-IPAAm is observed.

2.3 Films of 25 µm thickness are irradiated, discontinuously, under air using an Electrocurtain type electron accelerator operating at 150 KeV and a total dose of 100 Kgray. The irradiated films are immediately trapped in liquid nitrogen (during transport) and stored at very low temperature (freezer at -180 ° C) to preserve the stability of the peroxides / hydroperoxides functions generated in area. After thawing, the pre-irradiated films are immediately immersed in an aqueous solution of NIPAAm (10% by weight) containing ferrous chloride (0.1% by weight) which has been degassed beforehand for 30 minutes with nitrogen. This reducing agent allows to chemically decompose the oxidized functions on the surface of the film at sites initiating the radical polymerization of N-IPAAm. The temperature of the grafting solution is 37 ° C. The molar ratio monomer / reducing agent and reaction time have an effect on the grafting yield and thickness of deposit.

2.4 Films of 25 µ thickness, one of the two faces of which has been masked by aluminum foil, are irradiated under nitrogen and using a Van der Graaf type electron accelerator (10 Mev) and a dose total of 250 Kgray. Irradiated films are immediately immersed in an aqueous solution of NIPAAm (10% by weight) which has been degassed beforehand for 30 minutes with nitrogen. The temperature of the grafting solution is 60 ° C. Media selectively grafted onto one of the two faces has been obtained.

We can conclude from these different tests that the highest grafting yields, leading to a thickness of deposit of more than 5 nanometers, were obtained during the pre-irradiation step, with a total radiation dose of 250 Kgray (time 25 min), leaving the pre-activated films in contact with the monomer lasting at least 2 hours. When the grafting time is shorter, grafting yields, and therefore thicknesses weaker deposits (less than 5 nanometers) were obtained at the surface of the polystyrene film. Such surfaces only confer partially the properties and functionalities sought.

Claims (17)

Process for continuously obtaining two-dimensional (2D) supports endowed with particular functionalities for the culture of anchor-dependent cells (CAD), comprising at least the following succession of steps: continuous production of reels of polymer film with a thickness less than or equal to 35 µ and a density between 0.9 and 1.25 g / cm ³ from granules of a given polymer, activation of said polymer film by any means making it possible to generate reactive groups, in particular radicals and / or peroxide, hydroperoxide, or amine functions; a covalent grafting between the activated polymer film and a polymer, a copolymer or a macromolecule of interest whose functionality is sought, said grafting being obtained by immersion of the film in a monomer solution, the copolymerization being initiated by the free radicals created by activation under β radiation, the immersion time being directly correlated to the desired thickness of the polymer grafted onto the film; if necessary, washing to remove the monomers not consumed and not fixed on the film, followed by drying of the grafted film, cutting of the grafted polymer film by continuous punching, the size of the punches being chosen as a function of that of the desired 2D supports. The method of claim 1 wherein the polymer film has a contact angle  between 30 ° and 90 °. The method of claim 1 wherein the activation is performed by Corona discharge, plasma or electron bombardment. The method of claim 3 wherein the activation polymer film electronics is made by bombardment with a β radiation, under an inert atmosphere. The method of claims 1 to 4 wherein the thickness of the polymer layer grafted to the surface of the film is determined by the combination of the following parameters: the total dose of β irradiation received by the film, the concentration of monomer in the grafting bath, the grafting time, grafting temperature. Method according to one of the preceding claims, in which the polymer or copolymer of interest is chosen from poly-N-alkyl derivatives (meth) acrylamides, their respective copolymers, poly-N-acryloyl piperidine or poly-N-acryloyl pyrrolidine whose desired functionality is cryosensitivity. Process according to claims 1 to 5 in which the polymer or the copolymer of interest is a hydrophilic polymer of the polyoxide type PEO amino ethylene whose desired functionality is biocompatibility. Process according to Claims 1 to 5, in which the one or more macromolecule (s) of interest are one or more specific ligands of cellular receptors and whose desired functionality is adhesion selective for cells carrying this receptor (s). The method of claim 6 wherein when the film polymer (substrate) is made of polystyrene and when the grafted polymer is chosen from poly-N-alkyl (meth) acrylamide derivatives, their respective copolymers, poly-N-acryloyl-piperidine or poly-N-acryloyl-pyrrolidine, a thickness of 40 to 60 Å is obtained by the combination a total irradiation dose of 50 to 250 KGray, a time and a immersion temperature in the monomer solution, respectively 1 to 3 hours and 50 to 70 degrees. The method of claim 1 wherein the activation is prior to the grafting of the polymer or copolymer of interest, the functionality is sought. Two-dimensional (2D) microsupports with functionalities particular for the mass culture of anchor-dependent cells (CAD), as obtained by a process according to any one of previous claims, characterized in that the thickness of the film polymer is between 10 and 35 microns, and that of the polymer, covalently grafted copolymer or macromolecule of interest have a thickness between 1 and 10 nm. Microsupports according to claim 11 in which the grafted polymer is a cryosensitive polymer chosen from derivatives of poly-N-alkyl (meth) acrylamides, their respective copolymers, poly-N-acryloyl-piperidine or poly-N-acryloyl-pyrrolidine. Supports according to claim 11 in which the polymer grafted is a hydrophilic polymer of the polyethylene ethylene PEO type amine. Supports according to claim 11 in which the one or more grafted macromolecule (s) are specific ligand (s) cell receptors. Device for the continuous preparation of 2D-MS supports endowed with particular properties, prepared according to a process as described above and using a covalent fixing of a biological polymer, copolymer or macromolecules on a substrate made of a polymer film , said device comprising: a system for unwinding / winding the polymer film, the film being driven at a speed chosen at each of the stages: activation, grafting, punching; an electron accelerator equipped to activate film reels continuously; a container intended to contain the solution of monomers, polymers or macromolecules to be grafted, into which the polymer film passes continuously at a determined speed and driven by the winding / unwinding system; a cutting tool allowing the punching of the film during its unwinding. Device according to claim 15 in which the system of unwinding / winding of the film allows the advancement of said film to a speed between 0.05 and 8 m per minute. Device according to claim 15 or 16 wherein the tool for cutting consists of a tool block on which are inserted, in the length of the film, rows of circular punches in ceramic carbide, and the corresponding impression block.

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